Thromboxane A2 (TXA2) and its immediate precursor prostaglandin H2(PGH2) are potent platelet aggregating and vasoconstrictor substances. They appear to share a common receptor termed the TXA2/PGH2 receptor. Thromboxane A2 synthesis is increased in a variety of cardiovascular and renal diseases and has been implicated as an important pathophysiologic mediator in these diseases. In recent years, the presence of a TXA2/PGH2 receptor in human platelets has been demonstrated using radiolabelled ligand binding studies. The long range objectives of this laboratory are to determine the relationship between the structure and function of the human platelet TXA2/PGH2 receptor. To achieve these objectives the present proposal has as its specific aims to; 1) clone the human platelet TXA2/PGH2 receptor from the human erythroleukemia cell, a cell that contains the receptor and has platelet protein markers. The cDNA for the receptor will be used to deduce its primary amino acid sequence. 2) Determine the mechanism(s) of desensitization of the receptor, 3) purify the receptor using a combination of affinity chromatography, lectin chromatography and HPLC, 4) synthesize photoaffinity and affinity agonists and antagonists and ligands for the preparation of affinity columns for the biochemical characterization and purification of the receptor, 5) determine the factors that account for the differences between agonist and antagonist binding and 6) determine the characteristics of the various domains of the receptor and their relationship to the function of the receptor. These studies will utilize a combination of molecular biological, pharmacological, biochemical and immunological techniques and synthetic chemistry. Specifically, radioligand binding studies with an 125I-labelled agonist and antagonists, measures of platelet function, affinity and photoaffinity labeling with radiolabelled ligands, partial proteolysis and peptide mapping, cloning and antibodies directed at various amino acid sequences of the receptor will be used. Utilizing these approaches a better understanding of the relationship of structure to function of this important receptor should be forthcoming. This may ultimately lead to the development of specific TXA2/PGH2 receptor antagonists that could serve as useful therapeutic agents for the treatment of cardiovascular diseases.